The invention concerns a new process for the fermentative production of nourseothricin and its recovery in the form of its salts and adsorbates. Nourseothricin is an antibiotic belonging to the group of streptothricines, which after delivery in ergotropic doses with animal feed upon agricultural utilization effects an accelerated increase in living matter as well as simultaneously a decrease in feed expense. It is thereby of use for animal production as well as the pharmaceutical and mixed feed agent industries.
The antibiotic nourseothricin, isolated from the culture of a variety of streptomycae noursei ATCC 11455, is effective in vitro against Gram-positive and Gram-negative bacteria as well as against mycobacteria (Bradler, G. and H. Thrum: Nourseothricin A and B, two new antibacterial antibiotics of a Streptomycae-Noursei variation. Zschr. Allgem. Mikrobiol. 3, 105-112 (1963)). Nourseothricin is a base soluble in water and lower alcohols. Easily manageable forms are its salts. The molecule of nourseothricin is constructed from the amino sugar gulosamine and the amino acids streptolidine and .beta.-lysine. The individual components of nourseothricin distinguish by the number of peptide-like .beta.-lysine groups connected with one another in the molecule. The nourseothricin complex is composed of about 90% to approximately the same portions of both main components F and D as well about 10% of both by-components C and E (Grafe, U.; Bocker, H.; Reinhardt, G. und H. Thrum: Regulative Beeinflussung der Nourseothricinbiosynthese durch o-Aminobenzoesaure in Kulturen des Streptomyces noursei JA 3890b. Zschr. Allg. Mikrobiol. 14, 659-675 (1974)).
The Streptomycete stock forming the antibiotic Nourseothricin is placed under the designation Streptomyces noursei ZIMET JA 3890b in the Stammsammlung des Zentrallinstitute fur Mikrobiologie und experimentelle Therapie der Akademie der Wissenschaften der DDR and now carries the designation Streptomyces noursei ZIMET 43716.
According to the processes known from literature (Bocker, H. and H. Thrun: Stimulation of nourseothricin produced by aminobenzoie acids. In: Herold, M. and Z. Gabriel: Antibiotics--Advances in research, production and clinical use. London 1966; p. 584-587) the cultivation follows under aerobic conditions. For this purpose spore material of this Streptomyces stock, lyophilically dried in soil, is applied to suitable agar nutrient base. After 6-10 days incubation at 28.degree.-30.degree. C., the so grown sporulated mycelium grass is used for inocculation of liquid sterile nutrient media. The inocculation can also follow directly with a preserve of submersed mycelium of the nourseothricin-forming Streptomycete stock, lyophilically dried in gelatin. The liquid nutrient media for the submersed cultivation of the inocculation material contains as substrate carbon and nitrogen sources as well as inorganic salts. As carbon sources, glucose and/or glycerine are employed. As nitrogen sources, particularly soy meal, various amino acids and/or ammonium salts come into consideration. The most favorable acidity for the cultivation of the inocculation material at the start of cultivation lies in the range of pH 6.0 up to 6.7.
The incubation follows at temperatures from 28.degree. to 30.degree. C. over a time period of 24 to 48 hours. The so provided inocculation material serves for the inocculation of sterile liquid nutrient media for the main culture. Such nutrient media are composed of carbon and nitrogen sources as well as inorganic salts. As carbon sources, cornstarch and/or glucose are employed. As nitrogen sources, particularly soy meal, various amino acids and/or ammonium salts come into consideration. The most favorable acidity for the antibiotic formation at the start of fermentation lies in the range of pH 6.0 up to pH 7.5. The cultivation follows at temperatures of 26.degree.-32.degree. C., preferably from 28.degree. to 30.degree. C., up to 150 hours.
The submersed cultivation of Streptomyces noursei ZIMET JA 3890b follows in known manner as a so-called vibration culture, or instead in aerated fermenters, without dosing of substrate or regulation of the pH-value.
It is known (Bradler, G. and H. Thrum: Nourseothricin A und B, zwei neue antibakterielle Antibiotika einer Streptomyces-noursei-Variante. Zschr.Allgem.Mikrobiol. 3, 105-112 (1963)) that the relatively small nourseothricin formation in the original fermentation medium can be increased approximately 5 to 10 times through the addition of aminoarylcarboxylic acids, particularly of 5-10 mM o-aminobenzoic acid, added at the start to the main culture, under the known technical conditions (Bocker, H. and H. Thrum: Stimulation of nourseothricin production by aminobenzoic acids. In: Herold, M. and Z. Gabriel: Antibiotics--Advances in research, production and clinical use. London 1966, p. 584-587). It is known from tests (summarized and cited in Grafe, U.; Steudel, A.; Bocker, H. and H. Thrum: Regulative influence of o-aminobenzoic acid (OABA) on the biosynthesis of nourseothricin in cultures of Streptomyces noursei JA 3890b. V. Effect of OABA on cytochrom levels and amino acid transport. Zschr.Allgem.Mikrobiol. 30, 185-194 (1980)) that o-aminobenzoic acid and/or other aminoarylcarboxylic acids specifically regulate the formation of cytochrome of the a-type (cytochrome-oxydase) and thereby indirectly inhibits not only the amino acid transport from the nutrient medium into the mycelium but also the oxidative desamination of the amino acids in the cells of the nourseothricin-former. Hereby is avoided on the one hand the repression of the secondary material exchange by means of a cellular over-supply of nitrogen catabolites. On the other hand, it is in this manner prevented that amino acids of the medium, serving as precursors of the nourseothricin, are extensively consumed by the antibiotic-former during its growth phase. These amino acids are thereby better available to the secondary material exchange in the course of the nourseothricin formation, since preferably the inorganic nitrogen source (ammonium nitrogen) is used for the biomass formation.
The fermentative nourseothricin production using aminoarylcarboxylic acids, which to known extent indeed make possible a substantial increase in the fermentation yield, displays however disadvantages, particularly with regard to cost, sterilization and waste water problems, which oppose a large-scale technical employment.
It is moreover known that the biosynthesis of most secondary metabolites is negatively influenced by excessive phosphate (summarized in: Martin, J. F. and A. L. Demain: Control of antibiotic biosynthesis. Microbiol. Rev. 44, 230-251 (1980)), technical microbial fermentations for the production of secondary metabolites, for example antibiotics, are performed with phosphate concentrations that are sub-optimal for the growth of the former. As a rule, complex nutrient base components of plant and animal origin, such as starch, soy meal, corn spring water, molasses, meat extracts, among others, are employed for fermentations of technical scale for the recovery of secondary metabolites. Indeed according to construction and pretreatment, such complex nutrient base components possess different contents of total phosphate of different biological availability. A portion of the available phosphate is provided in the fermentation media as soluble phosphate. This fact substantially complicates the standardization of the nutrient media.
The starting concentrations of soluble or available phosphate have, however, a different significance for the fermentative yield of the desired secondary metabolite, since on the one hand a determined amount of phosphate is necessary for the growth of the producing microorganisms and, on the other hand, too high phosphate starting concentrations inhibit the secondary metabolite formation.
The patent DD 155239 claims the use of concentration of the phosphate ions contained in the culture medium as a regulating measure for stabilization of the cultivation process. The process refers, however, only to the fermentative recovery of biomass, whereby the nominal value of the concentration is adjusted within the range from 20 to 60 mg/l phosphorus. The use of this process is not known for the regulation of biosynthesis of secondary metabolites in microbial batch-cultures.
With the improvement of microbial production techniques for the production of secondary metabolites, the nutrient medium and the producing microorganisms are so adapted to one another by means of mutual modifications that a compromise between both opposing regulative influences of the phosphate takes place. In this manner the step-wise increase in efficiency is, however, time-expensive and cost-intensive.
It is known, moreover, that in addition to the regulative influence by means of the composition of the nutrient medium, an improvement in the fermentative yield can also be obtained by means of a control of the fermentation process, preferably as a real-time control (Sukatsch, D. A. and G. Nesemann: Automatische Parametererfassung bei industriellen Fermentationen. Chemie-Technik 6, 261-265 (1977)).
With the use of real-time control necessary for an effective fermentation performance, there is present the difficulty of having to modulate the continuously measurable global process parameters, stabilizing in the fermentation techniques, such as pH-value, pO.sub.2 -value, dosage rate, exhaust gas composition, heat formation, instead of first by means of mainly time-expensive chemical analysis, subsequently determined substrate concentrations as primary regulative process parameter.
With regard to the isolation of the nourseothricin, it is known that the active substance is adsorbed in suitable cation exchangers from the culture filtrate freed of mycelium, is subsequently eluted with dilute acids, and after neutralization, concentration of the eluate, repeated precipitation of the crude product in the system methanol/acetone, and drying, and then obtained as pure substance (Bradler, G. and H. Thrum: Nourseothricin A und B, zwei neue antibakterielle Antibiotika einer Streptomyces-noursei Variante. Zschr.Allgem.Mikrobiol. 3, 105-115 (1963)).